The present invention relates generally to novel protein molecules and to derivatives, analogues, chemical equivalents and mimetics thereof capable of modulating cellular activity and, in particular, modulating cellular activity via the modulation of signal transduction. More particularly, the present invention relates to human sphingosine kinase and to derivatives, analogues, chemical equivalents and mimetics thereof. The present invention also contemplates genetic sequences encoding said protein molecules and derivatives, analogues, chemical equivalents and mimetics thereof. The molecules of the present invention are useful in a range of therapeutic, prophylactic and diagnostic applications.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Sphingosine kinase is a key regulatory enzyme in a variety of cellular responses. Its activity can affect inflammation, apoptosis and cell proliferation, and thus it is an important target for therapeutic intervention.
Sphingosine-1-phosphate is known to be an important second messenger in signal transduction (Meyer et al., 1997). It is mitogenic in various cell types (Alessenko, 1998; Spiegel et al., 1998) and appears to trigger a diverse range of important regulatory pathways including; prevention of ceramide-induced apoptosis (Culliver et al., 1996), mobilisation of intracellular calcium by an IP3-independant pathway, stimulation of DNA synthesis, activation of mitogen-activated protein (MAP) kinase pathway, activation of phospholipase D, and regulation of cell motility (for reviews see Meyer et al., 1997; Spiegel et al., 1998; Igarashi., 1997). Recent studies (Xia et al., 1998) have shown that sphingosine-1-phosphate is an obligatory signaling intermediate in the inflammation response of vascular endothelial cells to tumor necrosis factors-xcex1(TNFxcex1). In spite of its obvious importance, very little is known of the mechanisms that control cellular sphingosine-1-phosphate levels. It is known that sphingosine-1-phosphate levels in the cell are mediated largely by its formation from sphingosine by sphingosine kinase, and to a lesser extent by its degradation by endoplasmic reticulum-associated sphingosine-1-phosphate lyase and sphingosine-1-phosphate phosphatase (Spiegel et al., 1998). Basal levels of sphingosine-1-phosphate in the cell are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents. This response appears correlated with an increase in sphingosine kinase activity in the cytosol and can be prevented by addition of the sphingosine kinase inhibitory molecules N,N-dimethylsphingosine and DL-threo-dihydrosphingosine. This indicates that sphingosine kinase is an important molecule responsible for regulating cellular sphingosine-1-phosphate levels. This places sphingosine kinase in a central and obligatory role in mediating the effects attributed to sphingosine-1-phosphate in the cell.
Accordingly, there is a need to identify and clone novel sphingosine kinase molecules to facilitate the progression towards the more sensitive control of intracellular signal transduction via, for example, the elucidation of the mechanism controlling the expression and enzymatic activity of sphingosine kinase thereby providing a platform for the development of interventional therapies to regulate the expression or activity of sphingosine kinase. In work leading up to the present invention the inventors have purified and cloned a novel sphingosine kinase molecule.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
One aspect of the present invention provides an isolated nucleic acid molecule or derivative or analogue thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a novel sphingosine kinase protein or a derivative or mimetic of said sphingosine kinase protein.
Another aspect of the present invention provides an isolated nucleic acid molecule or derivative or analogue thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a human sphingosine kinase protein or a derivative or mimetic of said sphingosine kinase protein.
Yet another aspect of the present invention provides a nucleic acid molecule or derivative or analogue thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding, an amino acid sequence substantially as set forth in SEQ ID NO:2 or a derivative or mimetic thereof or having at least about 45% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:2.
Still another aspect of the present invention contemplates a nucleic acid molecule or derivative or analogue thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:1 or a derivative thereof or capable of hybridising to SEQ ID NO:1 under low stringency conditions.
Still yet another aspect of the present invention contemplates a nucleic acid molecule or derivative or analogue thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:1 or a derivative thereof, or capable of hybridising to SEQ ID NO:1 under low stringency conditions and which encodes an amino acid sequence corresponding to an amino acid sequence set forth in SEQ ID NO:2 or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2.
A further aspect of the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:1.
Another further aspect of the present invention contemplates a genomic nucleic acid molecule or derivative or analogue thereof capable of hybridising to SEQ ID NO:1 or a derivative thereof under low stringency conditions at 42xc2x0 C.
Still another further aspect of the present invention provides a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:1 or a derivative or analogue thereof including a nucleotide sequence having similarity to SEQ ID NO:1.
Yet another further aspect of the present invention provides an amino acid sequence set forth in SEQ ID NO:2 or a derivative, analogue or chemical equivalent or mimetic thereof as defined above or a derivative or mimetic having an amino acid sequence of at least about 45% similarity to at least 10 contiguous amino acids in the amino acid sequence as set forth in SEQ ID NO:2 or a derivative or mimetic thereof.
Still yet another further aspect of the present invention is directed to an isolated protein selected from the list consisting of:
(i) A novel sphingosine kinase protein or a derivative, analogue, chemical equivalent or mimetic thereof.
(iii) A protein having an amino acid sequence substantially as set forth in SEQ ID NO:2 or a derivative or mimetic thereof or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or a derivative, analogue, chemical equivalent or mimetic of said protein.
(iv) A protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:1 or a derivative or analogue thereof or a sequence encoding an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or a derivative, analogue, chemical equivalent or mimetic of said protein.
(v) A protein encoded by a nucleic acid molecule capable of hybridising to the nucleotide sequence as set forth in SEQ ID NO:1 or a derivative or analogue thereof under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID NO:2 or a derivative or mimetic thereof or an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2
(ii) A human sphingosine kinase protein or a derivative, analogue, chemical equivalent or mimetic thereof.
(vi) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a hormodimeric form.
(vii) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a heterodimeric form.
Another aspect of the present invention contemplates a method of modulating activity of sphingosine kinase in a mammal, said method comprising administering to said mammal a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease sphingosine kinase activity.
Still another aspect of the present invention contemplates a method of modulating cellular functional activity in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a nucleotide sequence encoding sphingosine kinase or sufficient to modulate the activity of sphingosine kinase.
Yet another aspect of the present invention contemplates a method of modulating cellular functional activity in a mammal said method comprising administering to said mammal an effective amount of sphingosine kinase or sphingosine kinase.
Still yet another aspect of the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of sphingosine kinase or sufficient to modulate the activity of sphingosine kinase wherein said modulation results in modulation of cellular functional activity.
A further aspect of the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of sphingosine kinase or sphingosine kinase for a time and under conditions sufficient to modulate cellular functional activity.
Yet another further aspect of the present invention relates to the use of an agent capable of modulating the expression of sphingosine kinase or modulating the activity of sphingosine kinase in the manufacture of a medicament for the modulation of cellular functional activity.
A further aspect of the present invention relates to the use of sphingosine kinase or sphingosine kinase in the manufacture of a medicament for the modulation of cellular functional activity.
Still yet another aspect of the present invention relates to agents for use in modulating sphingosine kinase expression or sphingosine kinase activity wherein said modulation results in modulation of cellular functional activity.
Another aspect of the present invention relates to sphingosine kinase or sphingosine kinase for use in modulating cellular functional activity.
In a related aspect of the present invention, the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
In yet another further aspect the present invention contemplates a pharmaceutical composition comprising sphingosine kinase, sphingosine kinase or an agent capable of modulating sphingosine kinase expression or sphingosine kinase activity together with one or more pharmaceutically acceptable carriers and/or diluents. Sphingosine kinase, sphingosine kinase or said agent are referred to as the active ingredients.
Yet another aspect of the present invention contemplates a method for detecting sphingosine kinase or sphingosine kinase mRNA in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for sphingosine kinase or sphingosine kinase mRNA or its derivatives or homologous for a time and under conditions sufficient for an antibody-sphingosine kinase or antibody-sphingosine kinase mRNA complex to form, and then detecting said complex.
Single and three letter abbreviations used throughout the specification are defined in Table 1.